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Am J Physiol Renal Physiol 295: F478-F487, 2008. First published June 4, 2008; doi:10.1152/ajprenal.90226.2008
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{alpha}-Adducin mutations increase Na/K pump activity in renal cells by affecting constitutive endocytosis: implications for tubular Na reabsorption

Lucia Torielli,1,* Simona Tivodar,2,* Rosa Chiara Montella,2,* Roberto Iacone,3 Gloria Padoani,1 Paolo Tarsini,1 Ornella Russo,3 Daniela Sarnataro,2 Pasquale Strazzullo,3 Patrizia Ferrari,1 Giuseppe Bianchi,4 and Chiara Zurzolo2

1Prassis sigma tau Research Institute, Milan; 2Dipartimento di Biologia e Patologia Cellulare e Molecolare, 3Department of Clinical and Experimental Medicine, Federico II University of Naples, Naples; and 4Chair of Nephrology, Dialysis and Hypertension, Vita-Salute San Raffaele University, San Raffaele Hospital, Milan, Italy

Submitted 1 April 2008 ; accepted in final form 29 May 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Genetic variation in {alpha}-adducin cytoskeletal protein is implicated in the polymerization and bundling of actin and alteration of the Na/K pump, resulting in abnormal renal sodium transport and hypertension in Milan hypertensive rats and humans. To investigate the molecular involvement of {alpha}-adducin in controlling Na/K pump activity, wild-type or mutated rat and human {alpha}-adducin forms were, respectively, transfected into several renal cell lines. Through multiple experimental approaches (microscopy, enzymatic assays, coimmunoprecipitation), we showed that rat and human mutated forms increased Na/K pump activity and the number of pump units; moreover, both variants coimmunoprecipitate with Na/K pump. The increased Na/K pump activity was not due to changes in its basolateral localization, but to an alteration of Na/K pump residential time on the plasma membrane. Indeed, both rat and human mutated variants reduced constitutive Na/K pump endocytosis and similarly affected transferrin receptor trafficking and fluid-phase endocytosis. In fact, {alpha}-adducin was detected in clathrin-coated vesicles and coimmunoprecipitated with clathrin. These results indicate that adducin, besides its modulatory effects on actin cytoskeleton dynamics, might play a direct role in clathrin-dependent endocytosis. The constitutive reduction of the Na/K pump endocytic rate induced by mutated adducin variants may be relevant in Na-dependent hypertension.

hypertension; Na transport; clathrin; trafficking; immunoprecipitation

ADDUCINS ARE CYTOSKELETAL proteins that regulate assembly of the spectrin-actin subcortical membrane network (19). They participate in several cell functions as substrates for protein kinase A and C (PKA, PKC) and tyrosine kinase phosphorylation (14, 22, 31), belonging to calmodulin binding and myristoylated alanine-rich PKC substrate (MARCKS) protein families which are involved in calcium cycling control, cell-to-cell contact, signal transduction, and cell motility (1, 32).

Genetic polymorphism in the {alpha}-adducin gene is associated with blood pressure variations in the Milan hypertensive strain (F316Y) (2) and in a subgroup of essential hypertensive patients (G460W-S586C) (6, 9), thus making {alpha}-adducin one of the most interesting candidate genes for human essential hypertension. Indeed, a large number of recent publications demonstrate positive association and linkage of mutated adducin with hypertension and cardiovascular risk. The few negative or null findings may be ascribed to use of DNA markers in linkage studies mapping too distant from the adducin locus or observation made in general, mainly normotensive populations, as extensively discussed in Ref. 3.

Functional and biochemical studies performed in vitro and in vivo in rat and humans indicate that mutated variants affect blood pressure by influencing the overall cation transport in renal epithelia, through modulation of the Na/K pump (11, 12, 29, 33). In vitro, recombinant mutated adducin variants activate the Na-K-ATPase more than the wild-type forms, by increasing its affinity for ATP and accelerating the rate of conformational change (11). In cultured normal rat kidney (NRK) cells, transfection of rat mutated {alpha}-adducin (Y316) increases Na/K pump activity and integrin surface expression and thickens the actin stress fibers, compared with the wild-type variant (316F) (46). Considering the active role of adducin in cytoskeleton organization (19, 22) and the contribution of cytoskeleton to the correct targeting of the Na/K pump to the appropriate membrane compartment (30, 43), several mechanisms may be responsible for the increased renal Na/K pump activity associated with mutated adducin (11, 12, 29): 1) altered distribution of the Na/K pump between the luminal and basolateral renal membranes, 2) increased turnover rate of enzymatic Na/K pump activity, and 3) reduced constitutive endocytosis of the Na/K pump. In fact, it was recently reported that dopamine-stimulated Na/K pump endocytosis is blunted by mutated adducin, likely because of increased adaptor protein 2 (AP2) phosphorylation due to a deficient protein phosphatase (PPA2) activity (10).

The aim of the present work, therefore, is to verify whether human mutated adducin affects Na/K pump function as previously demonstrated for the rat variants (46) and to identify the possible mechanism (mislocalization and/or altered trafficking). We transfected normal and mutated {alpha}-adducin variants into three renal epithelial cell lines, chosen according to species origin and cellular and Na/K pump characteristics: 1) Madin-Darby canine kidney (MDCK) cells, that represent an optimal model of polarized renal cells, with high Na/K pump expression (15, 52), transfected with rat adducin variants; 2) human embryonic kidney (HEK293) cells, used for transfection with human adducin variants. Both MDCK and HEK cells also allowed measurement of the number of Na/K pump binding sites, due to their high affinity for the ligand ouabain (18); and 3) NRK-52E cells were transfected with rat adducins (46).

We found that the mutated variants of both rat and human adducing increased Na/K pump activity, induced a higher number of Na/K pump sites and a significant reduction of the constitutive pump endocytosis rate, compared with the wild-type forms. Moreover, we found that adducin was present in the clathrin-coated vesicle (CCV) compartment and associated with clathrin, further supporting a role of {alpha}-adducin in the regulation of Na/K pump trafficking.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell Culture

MDCK (ATCC), NRK-52E (ECAC), and HEK293 cells (ATCC) were cultured in DMEM (GIBCO Laboratories/Invitrogen, Carlsbad, CA), with 5% (NRK and MDCK) or 10% (HEK293) fetal bovine serum.

Adducin Vectors and Stable Cell Transfection

Vectors for nontagged rat {alpha}-adducins and hemagglutinin (HA)-tagged {alpha}-adducin variants (rat and human) were generated as already described (10, 46). Rat {alpha}-adducins (F316Y) were transfected into NRK-52E and MDCK cells via calcium phosphate coprecipitation methods (46, 52). Human {alpha}-adducins (G460W-S586C) were transfected into HEK293 using Lipofectamine (Invitrogen). Stable transfectants were selected by addition of G418 sulphate and subcultured by ring isolation.

Western Blot Analysis

Proteins were quantified in cells washed with ice-cold saline and lysed in 0.1 N NaOH plus protease cocktail (1:200; Sigma, St. Louis, MO). Equal protein amounts were run on SDS-PAGE gels, transferred to nitrocellulose membrane, and probed with the appropriate antibody. Detection was performed with enhanced chemiluminescence or by means of an Odyssey laser scanner equipment (LI-COR Biosciences, Lincoln, NE). Autoradiograms were scanned with Bio-Rad imaging densitometer (Bio-Rad Laboratories, Hercules, CA). Adducin quantification was obtained using a purified recombinant {alpha}-adducin-HA as standard curve. The anti-{alpha}-adducin antibodies (total adducin), both monoclonal and polyclonal (rabbit), were a gift from Dr. S. Salardi (Prassis). The monoclonal and polyclonal anti-HA antibodies (transfected adducin) were from Covance (Princeton, NJ). The monoclonal anti-Na-K-ATPase {alpha}-1 was purchased from Upstate Biotechnology (Lake Placid, NY). Other antibodies are indicated in the figure legends. When not indicated, chemicals were purchased from Sigma.

Na/K Pump Activity Assay

MDCK cells were seeded on Transwell Clear filter (0.4-µm-pore size; Costar, Corning, NY). Monolayer integrity was confirmed by checking transepithelial resistance (Millicell-ERS, Millipore, Billerica, MA). Other cells were plated on 12-well multiplates.

Na/K pump activity was measured in Na-loaded cells incubated either with 10 µM monensin or in the absence of external potassium. Preliminary experiments were performed to find the optimal monensin concentration (25, 27) and the appropriate incubation time in K-free medium (46) to load the cells with Na+ and reach maximal Na/K pump activity.

Na/K pump activity was measured as ouabain-sensitive 86Rb uptake (New England Nuclear-Perkin Elmer, Waltham, MA), in the presence or absence of 10 mM for rat cells or 1 mM ouabain for human and dog cells, as already described (5, 25, 27, 46). The cells were lysed for radioactivity counting and protein dosage. Ouabain-sensitive 86Rb uptake was expressed as the equivalent K transport in nanomoles per milligram of protein per hour.

Intracellular Na Content

Cell Na content was assessed in the cell lysate by atomic absorption spectroscopy (5) (SpectraAA 220, Varian, Palo Alto, CA) or by incubating overnight, in the presence of 22Na (New England Nuclear-Perkin Elmer), up to equilibrium (25), before and after Na loading performed.

For atomic absorption measurement, the cells were washed with ice-cold Na-free MgCl2 sucrose medium and lysed in double-distilled water. In the presence of 22Na, the cells were quickly washed in saline at room temperature and lysed for radioactivity counting and protein dosage. The amount was expressed as nanomoles of Na per milligram of protein.

Na/K Pump Site Determination

The number of external pump sites was determined by [3H]ouabain binding (20 Ci/mmol specific activity; New England Nuclear-Perkin Elmer) to the membrane as previously described (27), incubating the cells in K-free medium for 60 min at 37°C.

Immunofluorescence

MDCK were grown on Transwell filters, washed with saline containing 1 mM CaCl2 and 1 mM MgCl2, fixed with methanol at –20°C for 10 min, and permeabilized with methanol:acetone (1:1). Images were collected using laser scanning confocal microscope (Zeiss LSM 510, Oberkochen, Germany) equipped with a plan APO x63 oil-immersion (numerical aperture 1.4) objective lens.

Biotinylation Assay

MDCK were grown on Transwell filters, washed with saline, cooled on ice, and biotinylated with NH-LC Biotin (Pierce, Rockford, IL) at 4°C. Biotinylation (53) was done according to a modification of the standard method in which the reaction took place at pH 9.0 rather than 7.5 (15). Cells were lysed using buffer 1 (1% Triton X-100, 150 mM NaCl, 5 mM EDTA, 0.2% BSA, 20 mM tris, pH 8) for 20 min. Biotinylated Na-K-ATPase was precipitated with agarose beads conjugated with streptavidin (Pierce) and then revealed with specific antibody.

Na/K Pump Endocytosis

The constitutive endocytosis of the Na/K pump was evaluated in cultured cells by two methods: 1) measurement of the internalized biotinylated Na/K pump and 2) measurement of the decreasing surface Na/K pump activity after blocking the insertion of newly synthesized pumps by Brefeldin A (BFA).

Na/K pump biotinylation. MDCK cells were biotinylated as described (15, 53, 54), held at 37°C for indicated times, and processed for internalization assay (15, 54). The residual surface NH-S-S-biotin was removed by reductive cleavage at 4°C with glutathione. Biotinylated internalized Na/K pumps were immunoprecipitated with streptavidin agarose beads. Laemmli sample buffer without β-mercaptoethanol (nonreducing conditions) was added to the sample, run on SDS-PAGE gel, and revealed by Western blotting, with specific antibody.

BFA block. BFA is a fungal metabolite that inhibits the secretory pathway dissolving the Golgi complex, thus preventing insertion of new pumps into the plasma membrane. Although BFA has multiple targets in vesicular transport, it does not affect clathrin-mediated endocytosis (34, 38, 48). NRK or HEK cells were incubated with BFA (10 µM) at 37°C and the Na/K pump constitutive endocytosis rate was assessed by measuring the ouabain-sensitive 86Rb uptake or the number of Na/K pump sites during BFA treatment (from 30 to 120 min).

Internalization of Transferrin Receptor

NRK cells were serum-starved for 2 h before experiment, incubated for increasing periods with biotinylated transferrin (50 µM) at 20°C (41, 47), washed with saline, excess nonlabeled transferrin, and 3% BSA, in sequence. The filters were fixed in 4% paraformaldehyde, permeabilized with 2% saponin, and probed with [125I]streptavidin.

Fluid-Phase Endocytosis

HEK cells were incubated with culture medium + 1% BSA for 2 h before experiment, then a trace amount of [125I]BSA was added (42). After increasing periods at 37°C, the cells were rinsed three times with saline, lysed, and counted.

CCV Preparation

Preparation of CCV-enriched fraction from HEK was performed as described by Hammond and Verroust (17) and modified by Chibalin et al. (7), utilizing discontinuous sucrose gradient.

Coimmunoprecipitation

Na/K pump. MDCK were lysed in buffer 1 for 20 min, samples were immunoprecipitated overnight using anti-Na-K-ATPase antibody bound to protein A Sepharose beads (GE Healthcare), run on SDS-PAGE, and revealed by Western blotting using anti-Na-K-ATPase {alpha}1 subunit, anti-adducin, and anti-HA antibodies, after being stripped.

HA-adducin. The CCV fraction from HEK was prepared as described. After overnight incubation at 4°C in buffer 2 (0.1% Triton X-100, 0.1 M MES, 1 mM EGTA, 0.5 mM MgCl2, 0.2 mg/ml NaN3, pH 6.5), samples were immunoprecipitated using anti-HA monoclonal antibody bound to protein A Sepharose beads. The samples were heated at 60°C for 15 min in the presence of Laemmli sample buffer 2x, run on SDS-PAGE, and revealed by Western blotting using anti-clathrin antibody.

Statistical Analysis

Each experiment was performed at least five times for each cell clone. The data reported are means ± SE of the replicates. ANOVA followed by post hoc analysis, performed among clones transfected with the same adducin variant, did not show any statistically significant difference. Therefore, the statistical comparison between adducin variants was carried out by pooling all the replicates of each group of clones.

For time course experiments (internalization of transferrin receptor, BFA treatment, fluid-phase), a linear function was generated from each point set and the slope was calculated. Statistical analysis was performed on the means of the slopes. Unpaired t-test was used to evaluate between-group differences: P < 0.05 was considered statistically significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Both adducin variants, either from rat or human, resulted overexpressed by three to five times above endogenous protein level in the different stably transfected clones. Since the main objective of this study was to compare the effects induced by the two genetic variants of adducin on Na/K pump function, the investigation was carried out only on those clones showing similar adducin expression levels to exclude aspecific effects due to different overexpressions. For MDCK, two clones transfected either with normal (NT clones 22, 23: 3.8-fold total adducin compared with untransfected cells) or mutated (HT clones 3, 19: 3.6-fold total adducin) rat {alpha}-adducin were compared; for HEK cells, four clones transfected with either normal (clones 12, 13, 16, 19: 4.7 ± 0.6-fold total adducin compared with untransfected cells) or mutated human adducin (clones 28, 33, 35, 36: 4.1 ± 0.4-fold total adducin) were considered.

Mutant {alpha}-Adducins Increase Na/K Pump Function Through Protein-Protein Interaction

MDCK cells transfected with rat adducin. The influence of the two genetic variants of rat adducin on Na/K pump function and surface expression was investigated in MDCK cells, which allow measurement of ouabain binding sites and which are considered a good model of polarized tubular renal cells (15, 16, 52). We first investigated the influence of the selecting agent, geneticin, used in the transfection procedure, on Na/K pump activity of untransfected and mock-transfected cells. As shown in Table 1, mock-transfected MDCK cells exhibited a statistically significant 14% decrease of Na/K pump activity and a parallel 10% decrease in ouabain binding when compared with the untransfected cells, resulting in a similar K influx per single site. Overall, the overexpression of transfected rat {alpha}-adducin (both variants) was associated with an increase in pump activity and a similar or slightly reduced number of ouabain binding sites, leading to an increase in the K influx per single site compared with both untransfected and mock-transfected cells (Table 1). These data therefore indicate that the selection procedure may have interfered with Na/K pump membrane expression in MDCK, but also suggest an effect of rat adducin overexpression per se. However, in both mutated clones used in the experiment, Na/K pump activity was significantly greater (+16%, P < 0.01) than in the two normal adducin clones. Intracellular Na content was similar among the different clones (on average 1,100 nmol Na/mg protein, not shown). Cells expressing the mutated adducin also had a significantly higher number of ouabain binding sites (+14%, P < 0.01): therefore, pump activity per single site was similar in the mutated and wild-type clones, suggesting that the higher Na pump activity in the mutated clones was secondary to a greater abundance of Na pump units.


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  		Table 1. Measurements of Na/K pump activity and ouabain binding sites in MDCK clones expressing {alpha}-adducin variants

 
Since an alteration of the actin cytoskeleton structure was already described in the presence of mutated adducin (46), correct basolateral insertion of the Na/K pump was tested. In polarized MDCK renal cells, the Na/K pump is mainly sorted to the basolateral plasma membrane (more than 90%) (15, 16, 30, 52). Indirect immunofluorescence by confocal microscopy analysis of filter-grown MDCK showed that the cell membrane is clearly stained in a similar way in untransfected and transfected MDCK cells with both rat adducin variants. In both untransfected and transfected cells, the staining is almost exclusively present in the membranes at the bottom (basal) of the cells and absent at the top (apical; Fig. 1A) (52). To quantify Na/K pump distribution, the apical and basolateral domains were selectively biotinylated and biotinylated proteins were precipitated using agarose beads conjugated with streptavidin. The amount of the Na/K pump {alpha}1 subunit was evaluated by Western blot. Figure 1B shows the Na/K pump {alpha}1 subunit that mainly runs at apparent 105 kDa. The faint band at 90 kDa was an aspecific band or degradation product, already observed under similar conditions (52). As already observed with confocal microscopy, both normal and mutated rat {alpha}-adducin clones expressed the Na/K pump almost exclusively at the basolateral (BL) cell surface (97% normal adducin clones, 98% mutated adducin clones), while {alpha}1 was barely detectable in the apical (AP) membrane fractions (Fig. 1B) only in untransfected (WT) cells as a very faint band.


Figure 1
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  		Fig. 1. Steady-state plasma membrane distribution of Na/K pump in filter-grown Madin-Darby canine kidney (MDCK) clones expressing MHS or MNS rat adducin. Steady-state distribution of Na-K-ATPase {alpha}-subunit on Costar filter was analyzed by immunofluorescence followed by laser-scanning confocal microscopy (A) and biotinylation assay (B). A: confocal immunofluorescence demonstrating that the Na/K pump is basolateral in polarized MDCK cells [MDCK WT: untransfected cells; clone (CL) 23 NT and CL 22 NT: MDCK transfected with normal {alpha}-adducin; CL 19 HT and CL 3 HT: MDCK cells transfected with mutant {alpha}-adducin]. Serial confocal sections were collected from top to bottom of cell monolayer. B: untransfected cells (MDCK WT) and MDCK cells transfected with normal (clones 23 NT and 22 NT) or mutant rat {alpha}-adducin (clones 19 HT and 3 HT) were grown on filters and biotinylated from the apical (AP) or basolateral (BL) side. After cell lysis, biotinylated Na/K pump was immunoprecipitated with agarose beads conjugated with streptavidin, analyzed by SDS-PAGE, and revealed with a monoclonal antibody to the {alpha}-subunit of Na-K-ATPase (UBI). Values at left are molecular weight standards in kDa.

 
These data indicate that the overexpression of rat rather than canine {alpha}-adducin, either normal or mutated, did not affect localization of the Na/K pump.

Enhanced Na/K pump function, induced by mutant adducin variants, together with the previous demonstration of a functional interaction between adducin and Na/K pump in cell-free system (11), led us to verify whether a similar physical interaction could be detected in intact MDCK cells. The Na/K pump was immunoprecipitated (Fig. 2A) and {alpha}-adducin detected in endogenous form with anti-adducin antibody in untransfected cells and in all transfected clones (Fig. 2B), where exogenous adducin was also detected by anti-HA antibody (Fig. 2C).


Figure 2
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  		Fig. 2. Coimmunoprecipitation of Na-K-ATPase and {alpha}-adducin (normal and mutated forms). The pictures represent one of three similar experiments. Untransfected MDCK cells (MDCK WT), MDCK cells transfected with normal rat {alpha}-adducin (clones 23 NT and 22 NT), and MDCK cells transfected with mutant rat {alpha}-adducin (clones 19 HT and 3 HT) were grown on dishes, the cells were lysed, Na-K-ATPase was immunoprecipitated using a mAb toward the {alpha}-subunit of the pump (UBI) and revealed using anti-Na-K-ATPase ({alpha}1 subunit; A). B: polyclonal (rabbit) anti-{alpha}-adducin. C: monoclonal anti-HA-tag. The positions of Na-K-ATPase {alpha}1 subunit (***), {alpha}-adducin (**), and HA-adducin (*) are indicated. Values at right are molecular weight standards in kDa.

 
HEK Cells Transfected with Human Adducin

HEK cells represent one of the models of human tubular cells more widely used for gene transfection (review in Ref. 44). In this study, we investigated the effect of human adducin variants on Na/K pump activity and expression in human HEK cells. As Table 2 shows, all HEK clones expressing the mutated human variant (G460W, S586C) exhibited a significant increase of pump activity (+34%, P < 0.01) and number of external sites (+16%, P < 0.05) compared with wild-type transfected clones, as observed for MDCK (Table 1). The activity slightly exceeded the rise in surface expression, resulting in a +15% K influx per single site (not significant; Table 2). As with MDCK, the greater Na/K pump activity of the mutated clones was not secondary to a different Na load, since similar intracellular Na content was measured in all clones (on average, 550 nmol/mg protein, data not shown). Interestingly, in untransfected and mock-transfected HEK cell, the number of Na/K pump sites and the activity were similar to those of clones overexpressing normal adducin (Table 2), as previously observed in NRK cells (46). This indicates that in HEK cells, as opposed to MDCK, a fourfold increase in wild-type adducin expression did not influence Na/K pump activity and membrane expression, while only the presence of the mutated variant affected pump functions.


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  		Table 2. Measurements of Na/K pump activity and ouabain binding sites in HEK293 clones expressing {alpha}-adducin variants

 
In summary, these data confirm that both rat and human adducin mutated variants increased Na/K pump activity and site number without altering pump distribution and localization and this effect is associated with a direct in vivo interaction between adducin and the pump.

Mutant {alpha}-Adducins Reduce the Constitutive Endocytic Rate of the Na/K Pump

Among others, a possible mechanism underlying increased Na/K pump activity and the surface expression associated with adducin mutations may be a defect in the process of constitutive endocytosis of the pump. In fact, {alpha}-adducin was recently reported to modulate dopamine-stimulated Na/K pump endocytosis via CCV (10). A potential clathrin consensus motif is present on human {alpha}-adducin at position 339-DNLVLLNPE-347 (26). Therefore, the involvement of rat and human adducin variants in the constitutive endocytosis of the Na/K pump was evaluated in transfected cells by measuring the internalized biotinylated Na/K pumps in MDCK and the time-dependent reduction of Na/K pump activity after cell incubation with BFA in NRK and HEK.

Biotinylation experiments, as Fig. 3 reports, showed that under our nonreducing conditions, most of the internalized Na/K pump is detected as {alpha}+β dimers at ~160 kDa, while the alpha subunits alone run at ~105 kDa. As already observed for Na/K pump activity and number of sites, the overexpression of both rat adducin variants in MDCK cells also influenced Na/K pump internalization. In fact, as Fig. 3 demonstrates, Na/K pump internalization was faster in both transfected clones than in untransfected cells (WT). This finding is in keeping with the lower number of pump sites in the transfected clones, compared with untransfected MDCK (Table 1). However, the endocytic pump rate was much lower (about three times) in cells transfected with the mutated (Cl3 HT) than with the wild-type adducin variant (Cl22 NT; Fig. 3). The presence of the {alpha}+β subunit complex of the internalized Na/K pump confirmed that the mutated adducin variant reduced the endocytic rate of the membrane Na/K pump functional complex.


Figure 3
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  		Fig. 3. Endocytic assays of Na/K pump in filter-grown MDCK clones expressing normal or mutated rat {alpha}-adducin: biotinylation technique. Pictures represent one of four similar experiments. Untransfected (WT) and transfected with normal (Cl 22 NT) or with mutated {alpha}-adducin (Cl 3 HT) MDCK cells were grown on filters and biotinylated with S-S-biotin on the basolateral side. Then, the cells were incubated in cell culture medium at 37°C for 15 and 30 min. The noninternalized S-S-biotin at time 0, 15, or 30 min was reduced with glutathione solution. The cells were lysed and biotinylated Na/K pump was immunoprecipitated with agarose beads conjugated with streptavidin, analyzed by SDS-PAGE under nonreducing conditions, and revealed with a monoclonal antibody to the {alpha}-subunit of Na-K-ATPase (UBI). For each Western blot: lane 1: control, nonglutathione-treated cell lysate; lanes 2, 3, 4: glutathione-treated cells after incubation at 37°C. Values at right are molecular weight standards in kDa. The positions of {alpha}+β dimer and {alpha} monomer of Na/K pump are indicated on the left.

 
By BFA method, the kinetics of pump activity were monitored up to 2 h to avoid possible toxic effect, although it is documented that the BFA effect can be reverted even after 8 h of incubation (38). Pump activity in NRK clones, expressing normal rat adducin, was significantly lower already 30 min after BFA treatment, compared with cells expressing the mutated variant, thus indicating a slower pump internalization associated with rat mutated adducin (Fig. 4A). Similarly, Na/K pump internalization after BFA resulted significantly in reduced HEK cells expressing human mutated adducin, with linear kinetics (Fig. 4B). The slope (% decrease/h) was significantly higher in normal (–13.1 ± 2.3%) than in mutated (–4.6 ± 2.7%, P = 0.03) adducin expressing cells. These findings were further confirmed with [3H]ouabain binding experiments: ouabain binding sites disappeared from the cell surface at a faster rate in HEK cells expressing wild-type (slope values –5.91 ± 0.72%) than in those expressing the mutated variant (–2.6 ± 0.83%, P = 0.03) during BFA treatment.


Figure 4
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  		Fig. 4. Endocytic assays of Na/K pump in NRK (A) or HEK (B) clones expressing, respectively, rat or human normal or mutated {alpha}-adducin variants: Brefeldin A (BFA) technique. The cells were incubated with 10 mM BFA in cell culture medium at 37°C at different times. Then, ouabain-sensitive 86Rb uptake activity was measured, as described in MATERIALS AND METHODS. Data are expressed as percent of respective controls without BFA incubation ({square}) processed at same time. *P < 0.05, **P < 0.01, ***P < 0.001 paired t-test vs. respective normal or mutated adducin control without BFA incubation. A: NRK clones: {blacksquare}: normal adducin cells (n = 5 with 2 different clones). {blacktriangleup}: Mutated adducin cells (n = 6 with 2 different clones). B: HEK clones: {blacksquare}: normal adducin cells (n = 8 with 2 different clones). {blacktriangleup}: Mutated adducin cells (n = 8 with 2 different clones).

 
These data indicate that both rat and human mutated adducins reduce the rate of Na/K pump constitutive endocytosis.

Mutant {alpha}-Adducins Influence Membrane Trafficking

We investigated whether the lower internalization rate associated with the expression of the mutated adducin variants was selective for the Na/K pump or whether it involved other proteins. For this purpose, we analyzed the role of human and rat adducin variants in HEK and NRK transfected cells as homologous species systems. Overall endocytotic activity of the transfected cells was measured as fluid-phase uptake of [125I]BSA (42) in HEK cells. The expression of mutated human {alpha}-adducin markedly reduced the average slope of uptake (1.2 ± 0.22 cpm/min) compared with wild-type adducin cells (2.3 ± 0.09, P = 0.005; Fig. 5).


Figure 5
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  		Fig. 5. Endocytic assays for fluid phase in HEK clones expressing normal or mutated human {alpha}-adducin. HEK cells transfected with normal or mutant human {alpha}-adducin were grown on 12-well plates, and then incubated with [125I]BSA in cell culture medium at 37°C at different times. The cells were lysed and radioactivity was counted. {blacksquare}: Normal adducin cells (n = 4 with 2 different clones). {blacktriangleup}: Mutated adducin cells (n = 4 with 2 different clones).

 
Biotinylated transferrin/transferrin receptor complexes were internalized within a few minutes in NRK cells (Fig. 6, top), but in cells expressing rat mutated adducin, this process occurred at a lower rate (Fig. 6, bottom), resulting in a significantly reduced amount of biotinylated transferrin in normal [17.3 ± 2.9 average slope values in arbitrary unit (AU)·mg protein–1·min–1], rather than in mutated, adducin NRK cells (8.3 ± 1.6, P = 0.04).


Figure 6
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  		Fig. 6. Transferrin receptor internalization in NRK cells expressing normal or mutated rat {alpha}-adducin. Top: biotinylated transferrin internalization after 8-min uptake in normal (left) and mutated (right) rat {alpha}-adducin NRK cells. The cells were seeded onto coverslip, serum-starved for 3 h, and incubated with biotinylated transferrin, and then rinsed with saline solution, fixed in saline-4% paraformaldehyde, permeabilized with saline –1% BSA-0.2% Triton X-100 for 15 min, and probed with a mAb anti-biotin. Secondary antibody was a donkey Cy3-conjugated anti-mouse (Jackson ImmunoResearch Laboratories). The coverslips were mounted using Molecular Probe ProLong Gold antifade reagent and observed at x40 magnification. Bottom: time course of transferrin internalization in normal ({blacksquare}) and mutated ({blacktriangleup}) {alpha}-adducin transfected NRK cells. NRK cells were grown on filters and treated as above. Permeabilized cells were probed with 125I-streptavidin and counted. {blacksquare}: Normal adducin cells (n = 7 with 2 different clones). {blacktriangleup}: Mutated adducin cells (n = 7 with 2 different clones).

 
Therefore, these data suggest a generalized influence of the mutated adducin forms on membrane protein trafficking.

Presence of {alpha}-Adducin in the CCV

To verify whether {alpha}-adducin directly participates in the CCV-dependent endocytotic machinery, we used overexpressing human normal {alpha}-adducin HEK cells. In the CCV fractions, {alpha}-adducin was identified by anti-HA antibody in Western blot (Fig. 7A, right arrow). The band was quantified and adducin was found enriched by 8.5-fold [CCV lane vs. total homogenate (Homog lane)], comparable to the clathrin heavy chain, enriched five times, with respect to the whole cell lysate (Fig. 7A, left arrow). Other proteins involved in CCV-dependent endocytosis were present and enriched in this fraction, such as adaptor AP2 (4 times), casein kinase 2 (CK2; 4.4 times), and protein phosphatases PP2A (7.4 times; data not shown) (4). Moreover, HA-adducin coimmunoprecipitated with clathrin from CCV fractions in HEK cell expressing either the normal or mutated human adducin forms (Fig. 7B, lanes 1 and 3). The clathrin bands were clearly absent in control IgG immunoprecipitates (lanes 2 and 4). When the two adducin variants were compared, it appeared that a higher amount of clathrin coimmunoprecipitated with the mutated instead of with the normal variant. Although the immunoprecipitated clathrin bands seemed rather weak, probably because of the small amount of protein (~100 µg) obtained from CCV preparations as starting material, similar results were obtained in other experiments.


Figure 7
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  		Fig. 7. Adducin in clathrin-coated vesicle (CCV). Pictures represent one of three similar experiments. A: Western blot analysis of {alpha}-adducin present in CCV fraction from HEK cells transfected with normal human HA-tagged {alpha}-adducin. Blots are presented in pairs. Lanes on the left were loaded with 10 µg of CCV preparation; lanes on the right with 100 µg of cell homogenate and probed with mAb anti-HA (Covance) or mAb anti-clathrin heavy chain (CHC; Cymbus). B: coimmunoprecipitation of clathrin and adducin from CCV in HEK cells. HEK cells were grown on dishes and lysed, and {alpha}-adducin was immunoprecipitated with agarose beads conjugated to anti-HA monoclonal antibody (Covance), analyzed by SDS-PAGE and Western blot, and revealed with a monoclonal antibody to clathrin anti-CHC (Cymbus) or anti-HA. Nonimmune IgG-conjugated beads were used as control for lysates from cells expressing normal or mutated {alpha}-adducin. Lane 1: IP pellet of HA beads from normal adducin overexpressing cells. Lane 2: IP pellet of control nonimmune IgG beads from normal adducin overexpressing cells. Lane 3: IP pellet of HA beads from mutated adducin overexpressing cells. Lane 4: IP pellet of control nonimmune IgG beads from mutated adducin overexpressing cells. Lane 5: homogenate used as molecular weight standard for both clathrin and HA-adducin. Values on the right are molecular weight in kDa.

 
These data indicate, for the first time, that adducin participates in the scaffolding machinery of the CCV system, strongly suggesting that the mutation differently affects protein interaction.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study investigated the influence of wild-type and naturally occurring mutated forms of rat and human {alpha}-adducin (2, 6) on the function, localization, and trafficking of the Na/K pump, when expressed in recognized models of renal epithelial cells. Our data indicate that, irrespective of the species of origin (rat or human), mutated variants of {alpha}-adducin enhance Na/K pump activity and decrease its rate of constitutive endocytosis in several renal cell lines. Moreover, direct interaction of adducin with the Na/K pump and clathrin is demonstrated here in vivo, for the first time, further supporting the role of {alpha}-adducin in CCV-dependent endocytosis machinery.

These data integrate well with previous results (10) on the stimulatory effect of mutated adducin variants on Na/K pump activity in opossum (OK) renal cells. Efendiev et al. (10) demonstrated that in transiently transfected OK cells, in the absence of any stimulation, both rat and human mutated {alpha}-adducin increased Na/K pump activity, with respect to the wild-type variant. In this study, we showed a similar effect when the pump activity was studied under Na-loading conditions in stable transfected epithelial renal cell lines from different species (dog, human) in addition to what was previously reported in rat cells (46). The influence of Na-loading procedure on the adducin-dependent modulatory effect on Na/K pump was excluded, since intracellular Na was comparable among clones from the same cell line, overexpressing the adducin variants. This strongly supports a direct adducin-Na-K-ATPase interaction.

To our knowledge, a direct comparison of Na/K pump function between MDCK and HEK renal cells has not been previously reported. Here, we observed some differences in the absolute values of Na/K pump activity, ouabain binding sites, and K influx per single site (or turnover rate, when calculated as nmol ATP/min) between untransfected MDCK and HEK cells, which may be ascribed to intrinsic characteristics of the cell lines. While only scanty data are available on Na/K pump characteristics in HEK cells, MDCK have been already fully characterized. Some authors reported ouabain binding site in MDCK similar to ours (27). A rather low turnover rate in MDCK cells has been also previously reported (21). It may be speculated that ATP concentration in MDCK cells, but not in HEK, is limiting for Na-K-ATPase activity, in keeping with the previously demonstrated effect of adducin that increases Na-K-ATPase activity by enhancing the affinity for ATP (11). This may be supported by studies in HeLa and intact renal tubular cells, where pump activity changes as linear function of the ATP content (20, 40). As opposed to MDCK cells, HEK exhibit high K influx per site, but it is well known that HEK are very different from MDCK, as they are not polarized and do not form tight junctions (44).

The finding that mutated {alpha}-adducin variants influenced the activity and the surface expression of the Na/K pump in a similar qualitative way, regardless of the cell line used for transfection, is therefore particularly relevant.

Such a specific effect of the mutated variants of {alpha}-adducin does not seem related to an altered Na/K pump distribution between luminal and basolateral renal cell compartments, since similar pictures were observed either in untransfected and in MDCK clones transfected with the two variants (see Fig. 1).

The hypothesis that the mutated adducin variants might negatively affect the constitutive pump internalization rate seems the most likely. Here, we show that in MDCK, HEK, and NRK cells stably transfected with the mutated adducins, either from rat or human, constitutive pump endocytosis decreased compared with the wild-type variants. Our findings are consistent with the data from Efendiev et al. (10) that show an inhibitory effect exerted by mutated adducin on dopamine-induced Na/K pump endocytosis, when transiently transfected in OK cells. In addition, in our stable transfected clones, we observed an inhibitory effect of the mutated adducin variants on more general endocytotic processes (fluid-phase and transferrin receptor endocytosis). This effect may be directly related to actin cytoskeletal stiffness induced by the mutated forms of adducin (46), since a rigid cortical actin cytoskeleton exerts an inhibitory effect on membrane protein traffic (35, 45, 49). An involvement of MARKCS proteins was described in the regulation of endocytosis through modulation of cytoskeleton dynamics (36) and calpain action is involved in the dissolution of the cortical actin ring, allowing endocytic movement (37). Adducin belongs to the MARKCS protein family (1, 32) and is a substrate for calpain (14). It is also a substrate for PKA and PKC (14, 24, 31) and Rho-associated kinases (13, 23), thus suggesting a critical role for {alpha}-adducins in the treadmill machinery of the actin cytoskeletal-based cellular movements, including endo-exocytosis. In addition, our findings sustain the hypothesis of a direct participation of adducin in the CCV system. A role for adducin in modulating the phosphorylation process controlling AP2 function in CCV was already reported (10). In our study, for the first time, {alpha}-adducin was found enriched in the CCV fraction, together with other proteins involved in CCV biochemical machinery and coimmunoprecipitated with clathrin. Finally, it was recently reported the presence of phosphorylated {alpha}- and β-adducin in the brain synaptic terminals, where endocytotic processes are highly relevant (8). These data, together with the presence of a consensus clathrin box motif on {alpha}-adducin, entitle adducin to membership in the rapidly growing endocytotic accessory protein family (39).

The {alpha}-adducin gene has a very high homology (~94%) between rats and humans (28). Mutations, both in rats (F316Y) (2) and humans (G460W/S586C) (6, 9), although occurring at different positions, correlate with blood pressure variations in the Milan hypertensive rat strain (2, 12) and, in at least one subgroup of hypertensive subjects, strongly associate with renal Na handling in these patients, as discussed in detail elsewhere (3, 6, 9, 29).

The reduced constitutive endocytosis of the Na/K pump described in this paper, resulting in an increased surface pump expression, is consistent with higher tubular sodium reabsorption observed both in rat at renal level (3, 12) and in essential hypertensive patients carrying the 460W {alpha}-adducin allele (3, 9, 29). These findings, therefore, provide a molecular interpretation of both the increased surface expression and function of the Na/K pump observed in Milan hypertensive rats (12, 33) and the reduced slope of the pressure-natriuresis relationship observed in hypertensive patients carrying the {alpha}-adducin G460W variant (29). In fact, it was demonstrated that a normal pressure-natriuresis relationship is strictly mediated by an efficient shift of Na/K pump units from the basolateral membrane of the renal tubular cells to an intracellular pool (51), with impairment of this process resulting in an altered tubular sodium reabsorption and high blood pressure in rat models of hypertension (50).

In conclusion, the findings presented in this study provide a genetic molecular explanation for the constitutive increase of the tubular sodium reabsorption underlying hypertension in rats and humans carrying the {alpha}-adducin mutated genotype (3) and are consistent with the hypothesis that mutations in the {alpha}-adducin gene alter the capacity of tubular epithelial cells to transport sodium, thereby increasing blood pressure.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by grants from the Ministero dell'Università e della Ricerca Scientifica e Tecnologica to C. Zurzolo (Programmi di Ricerca di Interesse Nazionale 2004 and 2006) and to P. Strazzullo [Finanziamento Indipendente per la Ricerca di Base (FIRB) 2001]. R. C. Montella was a recipient of a fellowship from FIRB 2001 DM n. 30 12/02/2004.


   DISCLOSURES
 
G. Bianchi is an advisor of Sigma-Tau (Pomezia, Italy) and L. Torielli, G. Padoani, P. Tarsini, and P. Ferrari are employees of this pharmaceutical company. Other authors declare that they have no conflict of interest.


   ACKNOWLEDGMENTS
 
We thank Dr. M. Grazia Tripodi for the adducin constructs, Dr. S. Salardi for antibodies, and S. Pastore for recombinant adducin purification.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. Zurzolo, Dipartimento di Biologia e Patologia Cellulare e Molecolare, and to L. Torielli, Prassis sigma tau Research Institute, via Forlanini 3, Settimo Milanese, Milan, Italy (e-mails: zurzolo{at}unina.it and lucia.torielli{at}prassis.it)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* L. Torielli, S. Tivodar, and R. C. Montella contributed equally to this work. Back


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